Long distance retrograde signaling from nerve endings to the soma is required for Neurotrophin-mediated cell survival and the control of neuronal phenotype. Despite its great importance, fundamental questions about this signaling process, which involves formation and transport of signaling endosomes, remain unresolved. Some issues are highly contested, such as how persistent, long-distance signaling can be achieved by an endosome-based system. A potential resolution was provided recently by our lab that pointed to involvement of a novel, specialized endocytotic pathway for neurotrophins and their Trk receptors. Dissecting this newly identified Neurotrophin/Trk signaling pathway and its role in retrograde axonal signaling is the primary focus of this grant. At the heart of this signaling pathway is the novel protein Pincher (Pinocytic Chaperone) identified in our lab. Pincher mediates the primary retrograde signaling pathway for NGF and the other neurotrophin Trk receptors, but not other receptor tyrosine kinases. EGF for example, which unlike NGF does not mediate long-distance neuronal survival, utilizes the classic short-lived, clathrin-mediated endosomal pathway that is Pincher-independent. We plan to definitively show that the EGF and NGF signaling pathways are distinct at the level of endosomes and, further, to identify the Pincher-associated proteins that account for these differences. To achieve this goal we propose three specific aims: (1) Identify the molecular components, and their functional roles, in Pincher/TrkA endosomes in the cell soma (2) Identify the molecular blueprint of Pincher/Trk endosomes at distinct stages in the retrograde transport pathway (3) Determine the relative contributions of clathrin and Pincher associated proteins to neuronal survival. Our studies are directly relevant to both Down's syndrome and Alzheimer's disease, wherein retrograde trophic support is defective and/or restoration of such support can be therapeutic in preventing neuronal loss and dysfunction. An understanding of the mechanisms for trophic factor signaling and delivery as proposed is essential to the rational design of therapeutics for combating these and other related diseases.